Journal: iScience

Article Title: Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease

doi: 10.1016/j.isci.2021.103702

Figure Lengend Snippet: Neuronal and glial expression of 128QHtt results in transposable element derepression (A) Semiquantitative RT-PCR analysis to assess the induction levels of the HD transgenic construct by elav-Gal4. cDNA was prepared from total RNA purified from HD ( elav-G4>128QHtt ) and control ( elav-G4/+ ) head tissues. The constitutive gapdh was examined as an endogenous control. (B) qRT-PCR analysis of transposable element expression in larval and adult brains of flies expressing 128QHtt in neurons ( elav-G4>128QHtt ); adult brains were analyzed at both young (0–2 days) and aged (10–12 days) time points; transcript levels were normalized to rp49 and displayed as fold change relative to flies carrying the elav-Gal4 driver with no 128QHtt transgene ( elav-G4/+ ). (C) Western blot assay of gypsy envelope protein (ENV) expression in HD larval and adult brains. GIOTTO protein was used as a loading control. Result was expressed as means for at least three independent biological replicates (∗p < 0.05; one-sample t test). (D) qRT-PCR analysis of TE expression in larval brains and adult heads isolated from 0- to 2-day-old and 10- to 12-day-old flies expressing 128QHtt with the pan-glial repo-Gal4 driver ( repo-Gal4>128QHtt ). Transcript levels were normalized to gapdh and displayed as fold change relative to flies carrying the repo-Gal4 driver with no128QHtt transgene ( repo-G4/+ ). (B and D) Bar graph represents the mean ± SEM from at least three independent experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; unpaired t tests). Red dots indicate individual data points. The black horizontal line indicates the fold change control value, set to 1. See also .

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with the following antibodies diluted in TBST: mouse anti-ENV 8E7 (1:500 , kindly provided by J. Gall), rabbit anti-GIOTTO (1:10,000 ( )), mouse anti-elav (1:500, 9F8A9 DSHB) and mouse anti-repo (1:500, 8D12 DSHB).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Construct, Purification, Quantitative RT-PCR, Western Blot, Isolation

Journal: iScience

Article Title: Transposable element activation promotes neurodegeneration in a Drosophila model of Huntington's disease

doi: 10.1016/j.isci.2021.103702

Figure Lengend Snippet:

Article Snippet: The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with the following antibodies diluted in TBST: mouse anti-ENV 8E7 (1:500 , kindly provided by J. Gall), rabbit anti-GIOTTO (1:10,000 ( )), mouse anti-elav (1:500, 9F8A9 DSHB) and mouse anti-repo (1:500, 8D12 DSHB).

Techniques: Recombinant, Multiplex Assay, Gel Extraction, Picogreen Assay, SYBR Green Assay, Over Expression, Marker, CNV Assay, Software

Journal: EMBO Reports

Article Title: TDP 2 negatively regulates axon regeneration by inducing SUMO ylation of an Ets transcription factor

doi: 10.15252/embr.201847517

Figure Lengend Snippet: Relationship among MXL‐1, TDPT‐1, ETS‐4, CEBP‐1, and PKA in the regulation of axon injury‐induced svh‐2 expression. Interaction between ETS‐4 and TDPT‐1. COS‐7 cells were transfected with plasmids encoding T7‐TDPT‐1, HA‐ETS‐4 (WT), HA‐ETS‐4(S73E) (SE), and HA‐ETS‐4(S73A) (SA), as indicated. Whole‐cell extracts (WCE) and immunoprecipitated complexes obtained with anti‐T7 antibody (IP: T7) were analyzed by immunoblotting (IB). In vitro SUMOylation of ETS‐4. COS‐7 cells were transfected with plasmids encoding HA‐ETS‐4 (WT) and HA‐ETS‐4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti‐HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti‐SUMO‐2/3 and anti‐HA antibodies. Arrows mark the SUMOylated‐ETS‐4 bands. Schematic representation of the two putative consensus SUMOylation sites and domain structure in ETS‐4. Domains are shown as follows: a pointed domain (PNT; yellow) and an Ets DNA‐binding domain (ETS; blue). SUMOylation of ETS‐4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA‐ETS‐4 (WT), HA‐ETS‐4(K32A; K83A) (2KA), T7‐TDPT‐1, His‐SUMO‐1, His‐SUMO‐2, and MXL‐1‐Myc, as indicated. SUMO‐conjugated proteins were isolated by cobalt affinity chromatography. ETS‐4 was detected by immunoblotting with anti‐HA antibody. Whole‐cell extracts (WCE) were analyzed by immunoblotting (IB).

Article Snippet: The membranes were immunoblotted with anti‐HA antibody (mouse 16B12; BioLegend) or anti‐T7 antibody (mouse T7‐Tag; Merck; or rabbit PM022; MBL), and bound antibodies were visualized with horseradish peroxidase (HRP)‐conjugated antibodies against rabbit or mouse IgG using an HRP chemiluminescent substrate reagent kit (Novex ECL; Invitrogen).

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Isolation, Affinity Chromatography

Journal: EMBO Reports

Article Title: TDP 2 negatively regulates axon regeneration by inducing SUMO ylation of an Ets transcription factor

doi: 10.15252/embr.201847517

Figure Lengend Snippet: COS‐7 cells were transfected with plasmids encoding HA‐ETS‐4 and T7‐TDPT‐1, and cell lysates were immunoprecipitated with anti‐HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti‐SUMO‐2/3, anti‐HA, and anti‐T7 antibodies. Arrows mark the SUMOylated‐ETS‐4 bands.

Article Snippet: The membranes were immunoblotted with anti‐HA antibody (mouse 16B12; BioLegend) or anti‐T7 antibody (mouse T7‐Tag; Merck; or rabbit PM022; MBL), and bound antibodies were visualized with horseradish peroxidase (HRP)‐conjugated antibodies against rabbit or mouse IgG using an HRP chemiluminescent substrate reagent kit (Novex ECL; Invitrogen).

Techniques: Transfection, Immunoprecipitation, In Vitro, Western Blot